Original Research
Transfer of Dabigatran and Dabigatran Etexilate Mesylate Across the Dually Perfused Human Placenta Priya Bapat, BMSc, Reuven Kedar, MD, Angelika Lubetsky, BSc, Jeremy N. Matlow, Katarina Aleksa, PhD, Howard Berger, MD, and Gideon Koren, MD OBJECTIVE: To assess the transplacental pharmacokinetics at term of the oral thrombin inhibitor, dabigatran, and its prodrug, dabigatran etexilate mesylate, to estimate fetal drug exposure. METHODS: Placentae were obtained with informed consent after cesarean delivery of healthy term pregnancies in Toronto, Ontario, Canada. The transplacental transfer of dabigatran and dabigatran etexilate mesylate was separately assessed using the ex vivo dual perfusion of an isolated human placental cotyledon. Dabigatran, at a concentration of 35 ng/mL, was added to the maternal circulation at the start of the experimental phase. Maternal and fetal samples were taken throughout the preexperimental (1 hour) and experimental (3 hours) phases for measurement of dabigatran and markers of placental viability. Separate placenta perfusions with dabigatran etexilate mesylate were conducted at an initial maternal concentration of 3.5 ng/mL. Dabigatran and dabigatran etexilate mesylate were measured using liquid chromatography–tandem mass spectrometry. RESULTS: There was slower transfer of dabigatran compared with antipyrine from the maternal-to-fetal circulation, because the median fetal-to-maternal concentration From the Motherisk Program, Division of Clinical Pharmacology, the Hospital for Sick Children, the Departments of Pharmacology and Toxicology and Pediatrics, University of Toronto, and the Department of Obstetrics and Gynecology, St Michael’s Hospital, Toronto, Ontario, Canada. Supported by a grant from the Canadian Institutes for Health Research. Priya Bapat is supported by an Ontario Graduate Scholarship. Gideon Koren is the holder of the Ivey Chair in Molecular Toxicology, the Department of Medicine, University of Western Ontario. Corresponding author: Gideon Koren, MD, The Motherisk Program, Division of Clinical Pharmacology & Toxicology, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, M5G 1X8, Canada; e-mail: gkoren@ sickkids.ca. Financial Disclosure The authors did not report any potential conflicts of interest. © 2014 by The American College of Obstetricians and Gynecologists. Published by Lippincott Williams & Wilkins. ISSN: 0029-7844/14
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ratio was 0.33 (interquartile range 0.29–0.38) after 3 hours (n53). The prodrug, dabigatran etexilate mesylate, had limited placental transfer as characterized by a fetal-tomaternal ratio of 0.17 (interquartile range 0.15–0.17) after 3 hours (n53). Placental viability markers for all perfusions were within normal ranges. CONCLUSION: This report provides direct evidence of the transfer of dabigatran and its prodrug across the term human placenta from the mother to the fetus. From a clinical perspective, these data suggest that, pending further study, dabigatran should not be used for anticoagulation of pregnant women, because the drug may have an adverse effect on fetal blood coagulation. (Obstet Gynecol 2014;123:1256–61) DOI: 10.1097/AOG.0000000000000277
A
nticoagulant therapy often is prescribed in pregnancy for a variety of clinical indications. Common indications include: prophylaxis of venous thromboembolism in pregnancies at high or intermediate risk for thromboembolic disease,1 treatment of acute venous thromboembolism in pregnancy, management of pregnancy in women with artificial heart valves, and the prevention of pregnancy-related complications in women with inherited or acquired thrombophilias and hypercoagulable antibody syndrome.2 Pregnancy itself is a hypercoagulable state3–5; thus, effective treatment for these women is critical.6–9 The classic oral anticoagulant, warfarin, is contraindicated during pregnancy because it is associated with a severe embryopathy.10–12 For this reason, unfractionated heparin or low-molecular-weight heparins are currently the drug of choice for most women requiring anticoagulation therapy during pregnancy.2 However, these are administered subcutaneously, resulting in patient discomfort, financial burden, and low adherence. The need for effective and well-tolerated oral antithrombotic agents with more predictable
OBSTETRICS & GYNECOLOGY
pharmacokinetic profiles has led to the development of new agents that directly target thrombin.13,14 Recently, the U.S. Food and Drug Administration approved the direct thrombin inhibitor, dabigatran etexilate mesylate, to reduce the risk of stroke and systemic embolism in patients with nonvalvular atrial fibrillation.15 Dabigatran is orally administered as the prodrug, dabigatran etexilate mesylate,16 which is rapidly converted by a serum esterase.17 Unlike warfarin and heparin, direct thrombin inhibitors are able to inhibit both free and fibrin-bound thrombin,14,18 potentially enabling more effective inhibition of coagulation.19,20 Although dabigatran offers a few of advantages over existing anticoagulants, there are currently no data regarding its placental transfer.2,21 The aim of the present study was to assess the transplacental pharmacokinetics at term of dabigatran and its prodrug, dabigatran etexilate mesylate, by using the dualperfusion human placental model.
MATERIALS AND METHODS The dual perfusion of a single placental cotyledon was originally described by Miller et al22 and adapted in our laboratory.23,24 The present study was approved by the research ethics board at St Michael’s Hospital in Toronto, Ontario, Canada. Placentae were obtained with informed consent after cesarean delivery of uncomplicated term pregnancies. The placentae were transported to the on-site laboratory in ice-cold heparinized phosphate-buffered saline and examined for evidence of physical damage during delivery. A clearly defined artery–vein pair on the fetal side with minimal branching was chosen for cannulation, and independent maternal and fetal circulations were established within 30 minutes of delivery.23 Perfusate consisted of 10.9 g/L M199 tissue culture medium containing 40,000 molecular-weight dextran (maternal 7.5 g/L; fetal 30.0 g/L), glucose (1.0 g/L), heparin (2,000 units/L), and kanamycin (100 mg/L). Antipyrine (1 mM) is added to the maternal perfusate as a flow-dependent marker of passive diffusion.25 To mimic physiologic conditions in maternal and fetal blood,26 maternal and fetal perfusates were buffered to pH 7.4 and 7.35, respectively, by the addition of small volumes of sodium bicarbonate and hydrochloric acid. A single-perfusion experiment consisted of a 1-hour preexperimental control phase followed by a 3-hour experimental phase. During both phases, flow rates were maintained at 2–3 and 13–14 mL/min in the fetal and maternal circuits, respectively. The maternal perfusate was equilibrated with 95% O2
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and 5% CO2 and the fetal with 95% N2 and 5% CO2, and the temperature of the circuits and perfusion chamber was kept at 37°C. The fetal and maternal circulations were maintained until all residual blood was cleared out of the vessels. At this point, the maternal and fetal circuits were closed and replaced with 250 mL and 150 mL of fresh perfusate, respectively. To confirm tissue viability, maternal and fetal samples were taken every 15 minutes to analyze O2 and CO2 content, pH, glucose concentration, and lactate production using an on-site blood gas analyzer. Additional samples were taken from the maternal and fetal reservoirs every 15 minutes for analysis of human chorionic gonadotropin (hCG) secretion and antipyrine transfer. The integrity of the placenta was analyzed by monitoring fetal reservoir volume and fetal arterial inflow pressure. The perfusion was terminated if there was a loss in fetal reservoir volume greater than 4 mL/h or if fetal arterial inflow pressure deviated from 30 to 60 mmHg for an extended period of time. Before the experimental period began, the perfusates in the maternal and fetal reservoirs were replaced with fresh media, and the circulations were closed and recirculated. In a closed-circuit experiment, dabigatran or dabigatran etexilate mesylate was added to the maternal circulation at a therapeutic concentration of 35 ng/mL or 3.5 ng/mL, respectively. Samples were taken from the maternal and fetal reservoirs for analysis of dabigatran transfer, O2 and CO2 content, pH, glucose consumption, and lactate production every 10 minutes for the first half hour and then every 30 minutes until the end of the 3-hour experimental period. Additional samples were taken directly from the maternal and fetal reservoirs every 30 minutes for analysis of hCG secretion and antipyrine transfer. Perfusate samples were stored at 220°C until analysis. Antipyrine samples were assayed using an ultraviolet-visible recording spectrophotometer W-160 reading at 350 nm, and hCG samples were assayed using an enzyme-linked immunosorbent assay kit and a Biotek Synergy HT microplate reader at 450 nm. Our method for extraction of dabigatran and dabigatran etexilate mesylate from perfusate was derived from Blech et al27 and Delavenne et al.28 Briefly, 50 microliters of perfusate sample was added into a 1.5-mL polypropylene tube along with 41.7 microliters of 0.6 mg/mL of Dabigatran-d7 internal standard diluted in methanol:0.1 N HCl (90:10). A standard curve was also prepared at the following level: 0, 1, 5, 10, 25, and 50 ng by adding dabigatran (0.7 mg/mL) and dabigatran etexilate mesylate (1 mg/mL) and 10 microliters of the internal standard (0.6 mg/mL) with 900 microliters
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methanol:0.1 N HCl (90:10). All samples were vortexed and centrifuged at 13,4003g for 5 minutes at room temperature. The supernatant was transferred to a 2-mL vial and 10 microliters was injected on the ultrahighperformance liquid chromatography. The analysis of the samples was performed on an ABSciex QTRAP5500 and Shimadzu Nexera ultrahigh-performance liquid chromatography system. Chromatography ran at a flow rate of 300 microliters/min on a Kinetex XB-C18 column 5032.1 mm, 2.6 micron with a gradient starting at 93% A (2 mM ammonium formate in ddH2O with 0.2% formic acid) and 7% B (2 mM ammonium formate in acetonitrile with 0.2% formic acid) ramping to 90% B at 0.6 minutes followed by isocratic elution at 90% B for 2.7 minutes. The total run time was 5 minutes, including reequilibration at the initial conditions. This method allowed for baseline chromatographic resolution of the dabigatran and dabigatran etexilate mesylate. The mass spectrometer was operated in positive electrospray ionization mode with a source temperature of 500°C and an internal standard voltage setting of 5,000. Precursor-to-product ion mass transitions were established by standard infusions. Data were acquired by multiple reaction monitoring mode with mass transitions as follows: 472.1/289.2 m/z for dabigatran 1 (transition used for quantitation), 472.1/324.0 m/z for dabigatran 2, 472.1/306.1 m/z for dabigatran 3, and 478.2/292.0 m/z for dabigatran-d7 1 (retention time 0.47 minutes); and 628.2/289.1 m/z for dabigatran etexilate mesylate 1 (transition used for quantitation) and 628.2/434.2 m/z for dabigatran etexilate mesylate 2 (retention time 0.56 minutes). Data analysis and peak integration were performed using Analyst 1.6 software from ABSciex. Sample concentrations were calculated by plotting peak area ratios (analyte/internal standard) against calibration curves of extracted matrix spiked standards.
All data are presented as median and interquartile range unless stated otherwise and comparisons between preexperimental and experimental phases were analyzed using a Wilcoxon sign rank test for nonparametric data.
RESULTS A total of six cotyledons from different placentae was perfused with dabigatran or dabigatran etexilate mesylate. The median weight of the six perfused cotyledons was 18.90 g (interquartile range 12.26– 24.69). Maternal and fetal flow rates were maintained at 14.10 (interquartile range 13.98–14.38) and 2.62 (interquartile range 2.09–2.90) mL/min, respectively. Throughout the experiments, measurements of placental viability and metabolic capacity remained within normal ranges (Table 1). The fetal arterial inflow pressure slightly decreased from the precontrol to the experimental period, yet remained within normal physiologic ranges. The rates of placental hCG production, glucose consumption, and oxygen consumption and delivery did not vary significantly between precontrol and experimental periods. Lactate production was constant during precontrol and experimental periods. Antipyrine equalized between the maternal and fetal reservoirs with a fetal-to-maternal ratio of 0.87 (interquartile range 0.82–0.92) after 3 hours. The rate of antipyrine disappearance from the maternal circulation was equal to the rate of appearance in the fetal circulation with values of 0.030 (interquartile range 0.023–0.031) and 0.032 (interquartile range 0.027–0.039) micromoles/g/min, respectively (P5.34). During all perfusions, fetal volume loss was never greater than 4 mL/hour and pH values remained within physiologic ranges. After addition of dabigatran (35 ng/mL) into the maternal circulation (n53), the rate of disappearance from the maternal circulation was faster over the first 30 minutes than the remainder of the perfusion
Table 1. Measurements of Placental Viability and Metabolic Capacity During the Perfusion Experiments (n56) Precontrol
Experiment
P
43.3 (32.5–45.6) 36.8 (15.3–108.8) 0.27 (0.19–0.33)
34.0 (30.9–38.8) 25.0 (8.2–55.4) 0.21 (0.15–0.30)
.09 .10 .58
0.013 (0.011–0.015) 0.38 (0.27–0.53) 0.13 (0.10–0.23)
0.019 (0.016–0.030) 0.40 (0.34–0.74) 0.19 (0.12–0.36)
.19 .14 .21
Viability Parameter Fetal arterial inflow pressure (mmHg) hCG production (milli-international units/g/min) Glucose consumption (micromoles/g/min) Oxygen (micromoles/g/min) Transfer Delivery Consumption
hCG, human chorionic gonadotropin. Data are median (interquartile range) unless otherwise specified.
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(0.13 [interquartile range 0.10–0.13; and 0.05 [interquartile range 0.03–0.06] ng/mL/min, P,.05; Figure 1A). After 3 hours, the median fetal concentration of dabigatran was 4.96 ng/mL (interquartile range 4.54–7.08) and the median fetal-to-maternal ratio was 0.33 (interquartile range 0.29–0.38). The transfer of dabigatran across the placenta was compared with antipyrine by comparing median fetal-to-maternal ratios (Fig. 2A). Lines have been fitted through the first few data points, because transfer is expected to
only occur in the maternal-to-fetal direction from 0 to 60 minutes. The fetal-to-maternal ratio for dabigatran increased at a slower rate as compared with that of antipyrine (0.0011 [interquartile range 0.0009–0.0018] and 0.0052 [interquartile range 0.0041–0.0061] min21; P,.05). When dabigatran etexilate mesylate (3.5 ng/mL) was added to the maternal circulation (n53), there was limited transfer across the placenta, evidenced by a median fetal concentration of 0.19 ng/mL (interquartile range 0.17–0.25) after the 3-hour perfusion
Fig. 1. A. Dabigatran and B. dabigatran etexilate mesylate concentrations in maternal and fetal reservoirs during the 3-hour experimental phase of the placenta perfusion after dabigatran (35 ng/mL, n53) or dabigatran etexilate mesylate (3.5 ng/mL, n53) was added to the maternal circulation. Data are shown as median and interquartile range at each time point.
Fig. 2. Fetal-to-maternal ratios for antipyrine and A. dabigatran or B. dabigatran etexilate mesylate during the 3-hour experimental phase of the perfusion. Regression lines have been fitted through the first few points (0–60 minutes) and extended to 180 minutes. Data are shown as median and interquartile range at each time point.
Bapat. Dabigatran Transfer Across the Human Placenta. Obstet Gynecol 2014.
Bapat. Dabigatran Transfer Across the Human Placenta. Obstet Gynecol 2014.
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(Fig. 1B). The median fetal-to-maternal ratio of dabigatran etexilate mesylate was 0.17 (interquartile range 0.15–0.17), suggesting that this prodrug’s concentrations did not equalize across the placenta. Dabigatran concentrations were also measured in the maternal and fetal circulations, but levels were found to be negligible in these prodrug perfusions.
DISCUSSION The results of this perfusion study document that dabigatran crosses the term human placenta relatively slowly, reaching a fetal-to-maternal ratio of 0.33 after 3 hours. The selected dabigatran concentration of 35 ng/mL was within the therapeutic range but not high enough to theoretically cause drug saturation of the single placental lobule in our model. The fetal-tomaternal ratio is reported and it is expected that a similar fetal-to-maternal ratio would be achieved at higher therapeutic concentrations (150 ng/mL). Because dabigatran is administered as a prodrug, additional perfusions were conducted with the prodrug dabigatran etexilate mesylate. Dabigatran etexilate mesylate was found to have limited placental transfer, as evidenced by a fetal-to-maternal ratio of 0.16 after 3 hours. The results of the dabigatran perfusions are of greater clinical relevance, because this is the form that reaches the placenta through the maternal circulation, because dabigatran etexilate mesylate is rapidly cleaved to dabigatran through plasma esterase.17 Dabigatran is highly polar and exhibits hydrogen bonding, both of which are factors that can reduce transport across the placental barrier.29 The relatively large molecular weight of dabigatran (molecular weight 628 Da) likely also limited its transfer, because molecules above 600 Da cross the placental barrier less readily.30 In vivo studies have shown that P-glycoprotein may be involved in the efflux of dabigatran.30 Taken together, the physicochemical and pharmacokinetic properties of dabigatran may be able to explain its limited placental transfer shown in the placenta perfusions. The placenta expresses several efflux transporters, including P-glycoprotein and breast cancer resistance protein and thus plays a major role in determining fetal drug exposure. However, the role of placental transporters was not analyzed in the present study. Future studies with the placental perfusion model or with cell culture preparations can determine a potential role of placental P-glycoprotein in the disposition of dabigatran across the human placenta. A limitation of this model is that full-term placentae are used. As a result, it is not possible to extrapolate
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our results to earlier gestational ages when P-glycoprotein is at a higher placental expression. However, pregnancy is classically a state of hypercoagulability, and hemostatic changes become more pronounced as the pregnancy progresses.3–5 There is a fivefold increase in the risk of developing venous thromboembolism during pregnancy and postpartum as compared with nonpregnant women of childbearing age,31 suggesting that anticoagulant therapy may be required at later stages in pregnancy and closer to term. In conclusion, the results of this perfusion study demonstrate that dabigatran crosses the term human placenta to some extent. Future studies will need to explore the role of placental drug transporters, especially P-glycoprotein, in affecting this process. From a clinical perspective, these data suggest that, pending further study, dabigatran should not be used for anticoagulation of pregnant women, because the drug may have an adverse effect on fetal blood coagulation. REFERENCES 1. Royal College of Obstetricians and Gynaecologists. Reducing the risk of thrombosis and embolism during pregnancy and the puerperium. Green-top Guideline No. 37a. London (UK): Royal College of Obstetricians and Gynecologists. 2. Bates SM, Greer IA, Middeldorp S, Veenstra DL, Prabulos AM, Vandvik PO. VTE, thrombophilia, antithrombotic therapy, and pregnancy: Antithrombotic Therapy and Prevention of Thrombosis, 9th ed: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines. Chest 2012;141(suppl):e691S–736S. 3. Bremme KA. Haemostatic changes in pregnancy. Best Pract Res Clin Haematol 2003;16:153–68. 4. Dahlman T, Hellgren M, Blomback M. Changes in blood coagulation and fibrinolysis in the normal puerperium. Gynecol Obstet Invest 1985;20:37–44. 5. O’Riordan MN, Higgins JR. Haemostasis in normal and abnormal pregnancy. Best Pract Res Clin Obstet Gynaecol 2003;17: 385–96. 6. Che Yaakob CA, Dzarr AA, Ismail AA, Zuky Nik Lah NA, Ho JJ. Anticoagulant therapy for deep vein thrombosis (DVT) in pregnancy. The Cochrane Database of Systematic Reviews 2010, Issue 6. Art. No.: CD007801. DOI: 10.1002/14651858. CD007801.pub2. 7. Fogerty AE, Connors JM. Management of inherited thrombophilia in pregnancy. Curr Opin Endocrinol Diabetes Obes 2009;16:464–9. 8. Greer IA. Thrombosis in pregnancy: updates in diagnosis and management. Hematol Am Soc Hematol Educ Program 2012; 2012:203–7. 9. James AH. Pregnancy-associated thrombosis. Hematol Am Soc Hematol Educ Program 2009;277–85. 10. Chan WS, Anand S, Ginsberg JS. Anticoagulation of pregnant women with mechanical heart valves: a systematic review of the literature. Arch Intern Med 2000;160:191–6. 11. Hassouna A, Allam H. Anticoagulation of pregnant women with mechanical heart valve prosthesis: a systematic review of the literature (2000–2009). J Coagul Disord 2010;2:81–8.
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12. McLintock C. Anticoagulant choices in pregnant women with mechanical heart valves: balancing maternal and fetal risks—the difference the dose makes. Thromb Res 2013;131(suppl 1):S8–10.
22. Miller RK, Wier PJ, Maulik D, di Sant’Agnese PA. Human placenta in vitro: characterization during 12 h of dual perfusion. Contrib Gynecol Obstet 1985;13:77–84.
13. Bauer KA. Recent progress in anticoagulant therapy: oral direct inhibitors of thrombin and factor Xa. J Thromb Haemost 2011; 9(suppl 1):12–19.
23. Derewlany LO, Leeder JS, Kumar R, Radde IC, Knie B, Koren G. The transport of digoxin across the perfused human placental lobule. J Pharmacol Exp Ther 1991;256:1107–11.
14. Huntington JA, Baglin TP. Targeting thrombin—rational drug design from natural mechanisms. Trends Pharmacol Sci 2003; 24:589–95.
24. Pollex E, Lubetsky A, Koren G. The role of placental breast cancer resistance protein in the efflux of glyburide across the human placenta. Placenta 2008;29:743–7.
15. Hankey GJ, Eikelboom JW. Dabigatran etexilate: a new oral thrombin inhibitor. Circulation 2011;123:1436–50.
25. Schneider H, Panigel M, Dancis J. Transfer across the perfused human placenta of antipyrine, sodium and leucine. Am J Obstet Gynecol 1972;114:822–8.
16. Stangier J, Clemens A. Pharmacology, pharmacokinetics, and pharmacodynamics of dabigatran etexilate, an oral direct thrombin inhibitor. Clin Appl Thromb Hemost 2009;15(suppl 1):9S–16S. 17. Eisert WG, Hauel N, Stangier J, Wienen W, Clemens A, van Ryn J. Dabigatran: an oral novel potent reversible nonpeptide inhibitor of thrombin. Arterioscler Thromb Vasc Biol 2010;30:1885–9. 18. van Ryn JHN, Waldman L, Wienen W. Dabigatran inhibits both clot-bound and fluid-phase thrombin in vitro: comparison to heparin and hirudin [abstract 570]. Arterioscler Thromb Vasc Biol 2008;28:e136–7. 19. Weitz JI, Leslie B, Hudoba M. Thrombin binds to soluble fibrin degradation products where it is protected from inhibition by heparin-antithrombin but susceptible to inactivation by antithrombin-independent inhibitors. Circulation 1998;97:544–52. 20. Weitz JI, Hudoba M, Massel D, Maraganore J, Hirsh J. Clot-bound thrombin is protected from inhibition by heparinantithrombin III but is susceptible to inactivation by antithrombin III-independent inhibitors. J Clin Invest 1990;86:385–91. 21. Cutts BA, Dasgupta D, Hunt BJ. New directions in the diagnosis and treatment of pulmonary embolism in pregnancy. Am J Obstet Gynecol 2013;208:102–8.
26. Reynolds F, Knott C. Pharmacokinetics in pregnancy and placental drug transfer. Oxf Rev Reprod Biol 1989;11:389–449. 27. Blech S, Ebner T, Ludwig-Schwellinger E, Stangier J, Roth W. The metabolism and disposition of the oral direct thrombin inhibitor, dabigatran, in humans. Drug Metab Dispos 2008; 36:386–99. 28. Delavenne X, Moracchini J, Laporte S, Mismetti P, Basset T. UPLC MS/MS assay for routine quantification of dabigatran— a direct thrombin inhibitor—in human plasma. J Pharm Biomed Anal 2012;58:152–6. 29. Pacifici GM, Nottoli R. Placental transfer of drugs administered to the mother. Clin Pharmacokinet 1995;28:235–69. 30. Hartter S, Sennewald R, Nehmiz G, Reilly P. Oral bioavailability of dabigatran etexilate (Pradaxa(Ò)) after co-medication with verapamil in healthy subjects. Br J Clin Pharmacol 2013;75: 1053–62. 31. Pomp ER, Lenselink AM, Rosendaal FR, Doggen CJ. Pregnancy, the postpartum period and prothrombotic defects: risk of venous thrombosis in the MEGA study. J Thromb Haemost 2008;6:632–7.
Build Your Reference List at www.greenjournal.org The Green Journal’s web site now makes research easier. The full-text view of each article features an “Article Tools” box. Clicking on the link “Export to CitationManager” allows you to export the citation information to EndNote®, ProCite®, or ReferenceManager®. rev 7/2013
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Transfer of Dabigatran and Dabigatran Etexilate Mesylate Across the Dually Perfused Human Placenta Priya Bapat, BMSc, Reuven Kedar, MD, Angelika Lubetsky, BSc, Jeremy N. Matlow, Katarina Aleksa, PhD, Howard Berger, MD, and Gideon Koren, MD OBJECTIVE: To assess the transplacental pharmacokinetics at term of the oral thrombin inhibitor, dabigatran, and its prodrug, dabigatran etexilate mesylate, to estimate fetal drug exposure. METHODS: Placentae were obtained with informed consent after cesarean delivery of healthy term pregnancies in Toronto, Ontario, Canada. The transplacental transfer of dabigatran and dabigatran etexilate mesylate was separately assessed using the ex vivo dual perfusion of an isolated human placental cotyledon. Dabigatran, at a concentration of 35 ng/mL, was added to the maternal circulation at the start of the experimental phase. Maternal and fetal samples were taken throughout the preexperimental (1 hour) and experimental (3 hours) phases for measurement of dabigatran and markers of placental viability. Separate placenta perfusions with dabigatran etexilate mesylate were conducted at an initial maternal concentration of 3.5 ng/mL. Dabigatran and dabigatran etexilate mesylate were measured using liquid chromatography–tandem mass spectrometry. RESULTS: There was slower transfer of dabigatran compared with antipyrine from the maternal-to-fetal circulation, because the median fetal-to-maternal concentration From the Motherisk Program, Division of Clinical Pharmacology, the Hospital for Sick Children, the Departments of Pharmacology and Toxicology and Pediatrics, University of Toronto, and the Department of Obstetrics and Gynecology, St Michael’s Hospital, Toronto, Ontario, Canada. Supported by a grant from the Canadian Institutes for Health Research. Priya Bapat is supported by an Ontario Graduate Scholarship. Gideon Koren is the holder of the Ivey Chair in Molecular Toxicology, the Department of Medicine, University of Western Ontario. Corresponding author: Gideon Koren, MD, The Motherisk Program, Division of Clinical Pharmacology & Toxicology, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, M5G 1X8, Canada; e-mail: gkoren@ sickkids.ca. Financial Disclosure The authors did not report any potential conflicts of interest. © 2014 by The American College of Obstetricians and Gynecologists. Published by Lippincott Williams & Wilkins. ISSN: 0029-7844/14
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ratio was 0.33 (interquartile range 0.29–0.38) after 3 hours (n53). The prodrug, dabigatran etexilate mesylate, had limited placental transfer as characterized by a fetal-tomaternal ratio of 0.17 (interquartile range 0.15–0.17) after 3 hours (n53). Placental viability markers for all perfusions were within normal ranges. CONCLUSION: This report provides direct evidence of the transfer of dabigatran and its prodrug across the term human placenta from the mother to the fetus. From a clinical perspective, these data suggest that, pending further study, dabigatran should not be used for anticoagulation of pregnant women, because the drug may have an adverse effect on fetal blood coagulation. (Obstet Gynecol 2014;123:1256–61) DOI: 10.1097/AOG.0000000000000277
A
nticoagulant therapy often is prescribed in pregnancy for a variety of clinical indications. Common indications include: prophylaxis of venous thromboembolism in pregnancies at high or intermediate risk for thromboembolic disease,1 treatment of acute venous thromboembolism in pregnancy, management of pregnancy in women with artificial heart valves, and the prevention of pregnancy-related complications in women with inherited or acquired thrombophilias and hypercoagulable antibody syndrome.2 Pregnancy itself is a hypercoagulable state3–5; thus, effective treatment for these women is critical.6–9 The classic oral anticoagulant, warfarin, is contraindicated during pregnancy because it is associated with a severe embryopathy.10–12 For this reason, unfractionated heparin or low-molecular-weight heparins are currently the drug of choice for most women requiring anticoagulation therapy during pregnancy.2 However, these are administered subcutaneously, resulting in patient discomfort, financial burden, and low adherence. The need for effective and well-tolerated oral antithrombotic agents with more predictable
OBSTETRICS & GYNECOLOGY
pharmacokinetic profiles has led to the development of new agents that directly target thrombin.13,14 Recently, the U.S. Food and Drug Administration approved the direct thrombin inhibitor, dabigatran etexilate mesylate, to reduce the risk of stroke and systemic embolism in patients with nonvalvular atrial fibrillation.15 Dabigatran is orally administered as the prodrug, dabigatran etexilate mesylate,16 which is rapidly converted by a serum esterase.17 Unlike warfarin and heparin, direct thrombin inhibitors are able to inhibit both free and fibrin-bound thrombin,14,18 potentially enabling more effective inhibition of coagulation.19,20 Although dabigatran offers a few of advantages over existing anticoagulants, there are currently no data regarding its placental transfer.2,21 The aim of the present study was to assess the transplacental pharmacokinetics at term of dabigatran and its prodrug, dabigatran etexilate mesylate, by using the dualperfusion human placental model.
MATERIALS AND METHODS The dual perfusion of a single placental cotyledon was originally described by Miller et al22 and adapted in our laboratory.23,24 The present study was approved by the research ethics board at St Michael’s Hospital in Toronto, Ontario, Canada. Placentae were obtained with informed consent after cesarean delivery of uncomplicated term pregnancies. The placentae were transported to the on-site laboratory in ice-cold heparinized phosphate-buffered saline and examined for evidence of physical damage during delivery. A clearly defined artery–vein pair on the fetal side with minimal branching was chosen for cannulation, and independent maternal and fetal circulations were established within 30 minutes of delivery.23 Perfusate consisted of 10.9 g/L M199 tissue culture medium containing 40,000 molecular-weight dextran (maternal 7.5 g/L; fetal 30.0 g/L), glucose (1.0 g/L), heparin (2,000 units/L), and kanamycin (100 mg/L). Antipyrine (1 mM) is added to the maternal perfusate as a flow-dependent marker of passive diffusion.25 To mimic physiologic conditions in maternal and fetal blood,26 maternal and fetal perfusates were buffered to pH 7.4 and 7.35, respectively, by the addition of small volumes of sodium bicarbonate and hydrochloric acid. A single-perfusion experiment consisted of a 1-hour preexperimental control phase followed by a 3-hour experimental phase. During both phases, flow rates were maintained at 2–3 and 13–14 mL/min in the fetal and maternal circuits, respectively. The maternal perfusate was equilibrated with 95% O2
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and 5% CO2 and the fetal with 95% N2 and 5% CO2, and the temperature of the circuits and perfusion chamber was kept at 37°C. The fetal and maternal circulations were maintained until all residual blood was cleared out of the vessels. At this point, the maternal and fetal circuits were closed and replaced with 250 mL and 150 mL of fresh perfusate, respectively. To confirm tissue viability, maternal and fetal samples were taken every 15 minutes to analyze O2 and CO2 content, pH, glucose concentration, and lactate production using an on-site blood gas analyzer. Additional samples were taken from the maternal and fetal reservoirs every 15 minutes for analysis of human chorionic gonadotropin (hCG) secretion and antipyrine transfer. The integrity of the placenta was analyzed by monitoring fetal reservoir volume and fetal arterial inflow pressure. The perfusion was terminated if there was a loss in fetal reservoir volume greater than 4 mL/h or if fetal arterial inflow pressure deviated from 30 to 60 mmHg for an extended period of time. Before the experimental period began, the perfusates in the maternal and fetal reservoirs were replaced with fresh media, and the circulations were closed and recirculated. In a closed-circuit experiment, dabigatran or dabigatran etexilate mesylate was added to the maternal circulation at a therapeutic concentration of 35 ng/mL or 3.5 ng/mL, respectively. Samples were taken from the maternal and fetal reservoirs for analysis of dabigatran transfer, O2 and CO2 content, pH, glucose consumption, and lactate production every 10 minutes for the first half hour and then every 30 minutes until the end of the 3-hour experimental period. Additional samples were taken directly from the maternal and fetal reservoirs every 30 minutes for analysis of hCG secretion and antipyrine transfer. Perfusate samples were stored at 220°C until analysis. Antipyrine samples were assayed using an ultraviolet-visible recording spectrophotometer W-160 reading at 350 nm, and hCG samples were assayed using an enzyme-linked immunosorbent assay kit and a Biotek Synergy HT microplate reader at 450 nm. Our method for extraction of dabigatran and dabigatran etexilate mesylate from perfusate was derived from Blech et al27 and Delavenne et al.28 Briefly, 50 microliters of perfusate sample was added into a 1.5-mL polypropylene tube along with 41.7 microliters of 0.6 mg/mL of Dabigatran-d7 internal standard diluted in methanol:0.1 N HCl (90:10). A standard curve was also prepared at the following level: 0, 1, 5, 10, 25, and 50 ng by adding dabigatran (0.7 mg/mL) and dabigatran etexilate mesylate (1 mg/mL) and 10 microliters of the internal standard (0.6 mg/mL) with 900 microliters
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methanol:0.1 N HCl (90:10). All samples were vortexed and centrifuged at 13,4003g for 5 minutes at room temperature. The supernatant was transferred to a 2-mL vial and 10 microliters was injected on the ultrahighperformance liquid chromatography. The analysis of the samples was performed on an ABSciex QTRAP5500 and Shimadzu Nexera ultrahigh-performance liquid chromatography system. Chromatography ran at a flow rate of 300 microliters/min on a Kinetex XB-C18 column 5032.1 mm, 2.6 micron with a gradient starting at 93% A (2 mM ammonium formate in ddH2O with 0.2% formic acid) and 7% B (2 mM ammonium formate in acetonitrile with 0.2% formic acid) ramping to 90% B at 0.6 minutes followed by isocratic elution at 90% B for 2.7 minutes. The total run time was 5 minutes, including reequilibration at the initial conditions. This method allowed for baseline chromatographic resolution of the dabigatran and dabigatran etexilate mesylate. The mass spectrometer was operated in positive electrospray ionization mode with a source temperature of 500°C and an internal standard voltage setting of 5,000. Precursor-to-product ion mass transitions were established by standard infusions. Data were acquired by multiple reaction monitoring mode with mass transitions as follows: 472.1/289.2 m/z for dabigatran 1 (transition used for quantitation), 472.1/324.0 m/z for dabigatran 2, 472.1/306.1 m/z for dabigatran 3, and 478.2/292.0 m/z for dabigatran-d7 1 (retention time 0.47 minutes); and 628.2/289.1 m/z for dabigatran etexilate mesylate 1 (transition used for quantitation) and 628.2/434.2 m/z for dabigatran etexilate mesylate 2 (retention time 0.56 minutes). Data analysis and peak integration were performed using Analyst 1.6 software from ABSciex. Sample concentrations were calculated by plotting peak area ratios (analyte/internal standard) against calibration curves of extracted matrix spiked standards.
All data are presented as median and interquartile range unless stated otherwise and comparisons between preexperimental and experimental phases were analyzed using a Wilcoxon sign rank test for nonparametric data.
RESULTS A total of six cotyledons from different placentae was perfused with dabigatran or dabigatran etexilate mesylate. The median weight of the six perfused cotyledons was 18.90 g (interquartile range 12.26– 24.69). Maternal and fetal flow rates were maintained at 14.10 (interquartile range 13.98–14.38) and 2.62 (interquartile range 2.09–2.90) mL/min, respectively. Throughout the experiments, measurements of placental viability and metabolic capacity remained within normal ranges (Table 1). The fetal arterial inflow pressure slightly decreased from the precontrol to the experimental period, yet remained within normal physiologic ranges. The rates of placental hCG production, glucose consumption, and oxygen consumption and delivery did not vary significantly between precontrol and experimental periods. Lactate production was constant during precontrol and experimental periods. Antipyrine equalized between the maternal and fetal reservoirs with a fetal-to-maternal ratio of 0.87 (interquartile range 0.82–0.92) after 3 hours. The rate of antipyrine disappearance from the maternal circulation was equal to the rate of appearance in the fetal circulation with values of 0.030 (interquartile range 0.023–0.031) and 0.032 (interquartile range 0.027–0.039) micromoles/g/min, respectively (P5.34). During all perfusions, fetal volume loss was never greater than 4 mL/hour and pH values remained within physiologic ranges. After addition of dabigatran (35 ng/mL) into the maternal circulation (n53), the rate of disappearance from the maternal circulation was faster over the first 30 minutes than the remainder of the perfusion
Table 1. Measurements of Placental Viability and Metabolic Capacity During the Perfusion Experiments (n56) Precontrol
Experiment
P
43.3 (32.5–45.6) 36.8 (15.3–108.8) 0.27 (0.19–0.33)
34.0 (30.9–38.8) 25.0 (8.2–55.4) 0.21 (0.15–0.30)
.09 .10 .58
0.013 (0.011–0.015) 0.38 (0.27–0.53) 0.13 (0.10–0.23)
0.019 (0.016–0.030) 0.40 (0.34–0.74) 0.19 (0.12–0.36)
.19 .14 .21
Viability Parameter Fetal arterial inflow pressure (mmHg) hCG production (milli-international units/g/min) Glucose consumption (micromoles/g/min) Oxygen (micromoles/g/min) Transfer Delivery Consumption
hCG, human chorionic gonadotropin. Data are median (interquartile range) unless otherwise specified.
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(0.13 [interquartile range 0.10–0.13; and 0.05 [interquartile range 0.03–0.06] ng/mL/min, P,.05; Figure 1A). After 3 hours, the median fetal concentration of dabigatran was 4.96 ng/mL (interquartile range 4.54–7.08) and the median fetal-to-maternal ratio was 0.33 (interquartile range 0.29–0.38). The transfer of dabigatran across the placenta was compared with antipyrine by comparing median fetal-to-maternal ratios (Fig. 2A). Lines have been fitted through the first few data points, because transfer is expected to
only occur in the maternal-to-fetal direction from 0 to 60 minutes. The fetal-to-maternal ratio for dabigatran increased at a slower rate as compared with that of antipyrine (0.0011 [interquartile range 0.0009–0.0018] and 0.0052 [interquartile range 0.0041–0.0061] min21; P,.05). When dabigatran etexilate mesylate (3.5 ng/mL) was added to the maternal circulation (n53), there was limited transfer across the placenta, evidenced by a median fetal concentration of 0.19 ng/mL (interquartile range 0.17–0.25) after the 3-hour perfusion
Fig. 1. A. Dabigatran and B. dabigatran etexilate mesylate concentrations in maternal and fetal reservoirs during the 3-hour experimental phase of the placenta perfusion after dabigatran (35 ng/mL, n53) or dabigatran etexilate mesylate (3.5 ng/mL, n53) was added to the maternal circulation. Data are shown as median and interquartile range at each time point.
Fig. 2. Fetal-to-maternal ratios for antipyrine and A. dabigatran or B. dabigatran etexilate mesylate during the 3-hour experimental phase of the perfusion. Regression lines have been fitted through the first few points (0–60 minutes) and extended to 180 minutes. Data are shown as median and interquartile range at each time point.
Bapat. Dabigatran Transfer Across the Human Placenta. Obstet Gynecol 2014.
Bapat. Dabigatran Transfer Across the Human Placenta. Obstet Gynecol 2014.
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(Fig. 1B). The median fetal-to-maternal ratio of dabigatran etexilate mesylate was 0.17 (interquartile range 0.15–0.17), suggesting that this prodrug’s concentrations did not equalize across the placenta. Dabigatran concentrations were also measured in the maternal and fetal circulations, but levels were found to be negligible in these prodrug perfusions.
DISCUSSION The results of this perfusion study document that dabigatran crosses the term human placenta relatively slowly, reaching a fetal-to-maternal ratio of 0.33 after 3 hours. The selected dabigatran concentration of 35 ng/mL was within the therapeutic range but not high enough to theoretically cause drug saturation of the single placental lobule in our model. The fetal-tomaternal ratio is reported and it is expected that a similar fetal-to-maternal ratio would be achieved at higher therapeutic concentrations (150 ng/mL). Because dabigatran is administered as a prodrug, additional perfusions were conducted with the prodrug dabigatran etexilate mesylate. Dabigatran etexilate mesylate was found to have limited placental transfer, as evidenced by a fetal-to-maternal ratio of 0.16 after 3 hours. The results of the dabigatran perfusions are of greater clinical relevance, because this is the form that reaches the placenta through the maternal circulation, because dabigatran etexilate mesylate is rapidly cleaved to dabigatran through plasma esterase.17 Dabigatran is highly polar and exhibits hydrogen bonding, both of which are factors that can reduce transport across the placental barrier.29 The relatively large molecular weight of dabigatran (molecular weight 628 Da) likely also limited its transfer, because molecules above 600 Da cross the placental barrier less readily.30 In vivo studies have shown that P-glycoprotein may be involved in the efflux of dabigatran.30 Taken together, the physicochemical and pharmacokinetic properties of dabigatran may be able to explain its limited placental transfer shown in the placenta perfusions. The placenta expresses several efflux transporters, including P-glycoprotein and breast cancer resistance protein and thus plays a major role in determining fetal drug exposure. However, the role of placental transporters was not analyzed in the present study. Future studies with the placental perfusion model or with cell culture preparations can determine a potential role of placental P-glycoprotein in the disposition of dabigatran across the human placenta. A limitation of this model is that full-term placentae are used. As a result, it is not possible to extrapolate
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our results to earlier gestational ages when P-glycoprotein is at a higher placental expression. However, pregnancy is classically a state of hypercoagulability, and hemostatic changes become more pronounced as the pregnancy progresses.3–5 There is a fivefold increase in the risk of developing venous thromboembolism during pregnancy and postpartum as compared with nonpregnant women of childbearing age,31 suggesting that anticoagulant therapy may be required at later stages in pregnancy and closer to term. In conclusion, the results of this perfusion study demonstrate that dabigatran crosses the term human placenta to some extent. Future studies will need to explore the role of placental drug transporters, especially P-glycoprotein, in affecting this process. From a clinical perspective, these data suggest that, pending further study, dabigatran should not be used for anticoagulation of pregnant women, because the drug may have an adverse effect on fetal blood coagulation. REFERENCES 1. Royal College of Obstetricians and Gynaecologists. Reducing the risk of thrombosis and embolism during pregnancy and the puerperium. Green-top Guideline No. 37a. London (UK): Royal College of Obstetricians and Gynecologists. 2. Bates SM, Greer IA, Middeldorp S, Veenstra DL, Prabulos AM, Vandvik PO. VTE, thrombophilia, antithrombotic therapy, and pregnancy: Antithrombotic Therapy and Prevention of Thrombosis, 9th ed: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines. Chest 2012;141(suppl):e691S–736S. 3. Bremme KA. Haemostatic changes in pregnancy. Best Pract Res Clin Haematol 2003;16:153–68. 4. Dahlman T, Hellgren M, Blomback M. Changes in blood coagulation and fibrinolysis in the normal puerperium. Gynecol Obstet Invest 1985;20:37–44. 5. O’Riordan MN, Higgins JR. Haemostasis in normal and abnormal pregnancy. Best Pract Res Clin Obstet Gynaecol 2003;17: 385–96. 6. Che Yaakob CA, Dzarr AA, Ismail AA, Zuky Nik Lah NA, Ho JJ. Anticoagulant therapy for deep vein thrombosis (DVT) in pregnancy. The Cochrane Database of Systematic Reviews 2010, Issue 6. Art. No.: CD007801. DOI: 10.1002/14651858. CD007801.pub2. 7. Fogerty AE, Connors JM. Management of inherited thrombophilia in pregnancy. Curr Opin Endocrinol Diabetes Obes 2009;16:464–9. 8. Greer IA. Thrombosis in pregnancy: updates in diagnosis and management. Hematol Am Soc Hematol Educ Program 2012; 2012:203–7. 9. James AH. Pregnancy-associated thrombosis. Hematol Am Soc Hematol Educ Program 2009;277–85. 10. Chan WS, Anand S, Ginsberg JS. Anticoagulation of pregnant women with mechanical heart valves: a systematic review of the literature. Arch Intern Med 2000;160:191–6. 11. Hassouna A, Allam H. Anticoagulation of pregnant women with mechanical heart valve prosthesis: a systematic review of the literature (2000–2009). J Coagul Disord 2010;2:81–8.
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23. Derewlany LO, Leeder JS, Kumar R, Radde IC, Knie B, Koren G. The transport of digoxin across the perfused human placental lobule. J Pharmacol Exp Ther 1991;256:1107–11.
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24. Pollex E, Lubetsky A, Koren G. The role of placental breast cancer resistance protein in the efflux of glyburide across the human placenta. Placenta 2008;29:743–7.
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16. Stangier J, Clemens A. Pharmacology, pharmacokinetics, and pharmacodynamics of dabigatran etexilate, an oral direct thrombin inhibitor. Clin Appl Thromb Hemost 2009;15(suppl 1):9S–16S. 17. Eisert WG, Hauel N, Stangier J, Wienen W, Clemens A, van Ryn J. Dabigatran: an oral novel potent reversible nonpeptide inhibitor of thrombin. Arterioscler Thromb Vasc Biol 2010;30:1885–9. 18. van Ryn JHN, Waldman L, Wienen W. Dabigatran inhibits both clot-bound and fluid-phase thrombin in vitro: comparison to heparin and hirudin [abstract 570]. Arterioscler Thromb Vasc Biol 2008;28:e136–7. 19. Weitz JI, Leslie B, Hudoba M. Thrombin binds to soluble fibrin degradation products where it is protected from inhibition by heparin-antithrombin but susceptible to inactivation by antithrombin-independent inhibitors. Circulation 1998;97:544–52. 20. Weitz JI, Hudoba M, Massel D, Maraganore J, Hirsh J. Clot-bound thrombin is protected from inhibition by heparinantithrombin III but is susceptible to inactivation by antithrombin III-independent inhibitors. J Clin Invest 1990;86:385–91. 21. Cutts BA, Dasgupta D, Hunt BJ. New directions in the diagnosis and treatment of pulmonary embolism in pregnancy. Am J Obstet Gynecol 2013;208:102–8.
26. Reynolds F, Knott C. Pharmacokinetics in pregnancy and placental drug transfer. Oxf Rev Reprod Biol 1989;11:389–449. 27. Blech S, Ebner T, Ludwig-Schwellinger E, Stangier J, Roth W. The metabolism and disposition of the oral direct thrombin inhibitor, dabigatran, in humans. Drug Metab Dispos 2008; 36:386–99. 28. Delavenne X, Moracchini J, Laporte S, Mismetti P, Basset T. UPLC MS/MS assay for routine quantification of dabigatran— a direct thrombin inhibitor—in human plasma. J Pharm Biomed Anal 2012;58:152–6. 29. Pacifici GM, Nottoli R. Placental transfer of drugs administered to the mother. Clin Pharmacokinet 1995;28:235–69. 30. Hartter S, Sennewald R, Nehmiz G, Reilly P. Oral bioavailability of dabigatran etexilate (Pradaxa(Ò)) after co-medication with verapamil in healthy subjects. Br J Clin Pharmacol 2013;75: 1053–62. 31. Pomp ER, Lenselink AM, Rosendaal FR, Doggen CJ. Pregnancy, the postpartum period and prothrombotic defects: risk of venous thrombosis in the MEGA study. J Thromb Haemost 2008;6:632–7.
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